Virus Purification Methods

ABSTRACT

The invention provides a method for the purification of a virus from a host cell, the method comprising the steps of: a) culturing host cells, b) infecting the host cells with a virus, c) treating the cell culture with nuclease, d) lysing the host cells to provide a lysate comprising the virus. The virus is preferably a recombinant adenovirus. The invention further provides a method for the purification of a recombinant virus expressing a heterologous protein that is capable of binding nucleic acid, comprising the steps of: a) culturing host cells, b) infecting the host cells with the recombinant virus, c) lysing the host cells to provide a lysate comprising the recombinant virus, d) subjecting the recombinant virus to anion exchange chromatography and size exclusion chromatography, characterized in that the virus-containing mixture is buffer exchanged at least once with a solution comprising at least 2 M NaCl, or another salt providing an equivalent ionic strength.

FIELD OF THE INVENTION

The invention belongs to the field of purification of virus, more inparticular recombinant adenovirus, from host cells.

BACKGROUND OF THE INVENTION

Viruses, either those occurring in nature, or recombinant versionsthereof, are used for vaccination and in the field of gene therapy. Itis possible for many viruses or virus-like particles to safely andefficiently propagate these in host cells (see for instance WO 01/38362,which describes the propagation of various viruses in host cells beingE1-immortalized retina cells). Recombinant adenoviruses are a preferredclass of viral vectors for use in gene therapy and for vaccinationpurposes. Such recombinant adenoviruses are usually deficient in atleast the E1 region, and are propagated in complementing cells providingthe E1-region, such as 293 cells, or E1-immortalized retina cells suchas PER.C6™ cells (see for instance U.S. Pat. No. 5,994,128).

After propagation of the viruses in the host cells, for virtually allapplications it is necessary to purify the viruses from the host cells,before further use.

International patent application WO 98/22588 describes methods for theproduction and purification of adenoviral vectors. The methods comprisegrowing host cells, infecting the host cells with adenovirus, harvestingand lysing the host cells, concentrating the crude lysate, exchangingthe buffer of the crude lysate, treating the lysate with nuclease, andfurther purifying the virus using chromatography.

Several other publications describe the purification of viruses fromhost cells, mostly concentrating on the use of specific chromatographicmatrices for purification of the virus from a host cell lysate, see e.g.U.S. Pat. Nos. 6,008,036, 6,586,226, 5,837,520, 6,261,823, 6,537,793,and international patent applications WO 00/50573, WO 02/44348 and WO03/078592.

Most of the described methods apply a nuclease treatment step to degradeDNA impurities. Despite the description of several processes regardingdifferent chromatography matrices, a need remains for alternative andpreferably improved methods for virus purification from host cellcultures. The present invention provides such methods.

DESCRIPTION OF THE FIGURES

FIG. 1. Scheme of the known method of harvesting the cells (T/B) versusthe method according to the invention (B/T), see example 1. T: Triton,B: Benzonase. p.i.: post infection.

FIG. 2. Host cell protein removal at clarification after T/B vs. B/Tprocess (see FIG. 1 for scheme). A silver-stained SDS-PAGE (4-12%bis-tris NuPAGE, Invitrogen) analysis of in process samples of 5separate purifications is shown (see example 1 and Table 1 for samples).Panel 2 is from a T/B harvest, wherein lysis preceded nuclease addition;panels 3-7 are from a B/T harvest, wherein nuclease was added beforelysis. The harvest (lanes 1) was clarified by a 0.5 μm Clarigard filter(lanes 2), followed by a 0.8/0.45 μm Sartopore 2 filter (lanes 3). M:marker, M_(w) in kD is shown alongside.

FIG. 3. Diafiltration with high salt removes histones during process(see example 2). A silver-stained SDS-PAGE is shown.

A. Permeate. Samples: 1: initial permeate. 2: after 4× concentration. 3:1^(st) DFV 0.3 M NaCl. 4: 3^(rd) DFV 0.6 M NaCl. 5: 4^(th) DFV 0.6 MNaCl. 6: 5^(th) DFV 1.0 M NaCl. 7: 6^(th) DFV 1.0 M NaCl. 8: 7^(th) DFV0.3 M NaCl. 9: 9^(th) DFV 0.3 M NaCl. M: marker, M_(w) in kD is shownalongside.

B. Retentate. Samples: 1: start sample. 2: after 4× concentration. 3:1^(st) DFV 0.3 M NaCl. 4: 2^(nd) DFV 0.6 M NaCl. 5: 6^(th) DFV 0.6 MNaCl. 6: 7^(th) DFV 1.0 M NaCl. 7: 8^(th) DFV 1.0 M NaCl. 8: 9^(th) DFV0.3 M NaCl. 9: 9^(th) DFV millex (0.22 μm filtrate of sample 8). M:marker, M_(w) in kD is shown alongside.

FIG. 4. Scheme of a preferred process according to the invention (seeexample 1).

FIG. 5. Removal of Ebola nucleoprotein (NP) from recombinant viruspreparations (see example 3, experiment 3.1 for details). Asilver-stained SDS-PAGE (4-12% bis-tris NuPAGE, Invitrogen) is shown. A:starting material. B: incubation with 1% Tween 20. C: incubation with2.5 M NaCl. The arrow denotes NP.

FIG. 6. Experiment for removal of Ebola nucleoprotein from recombinantvirus preparations (see example 3, experiment 3.3 for details).

FIG. 7. Non-reduced SDS-PAGE (panel 1) and Western blot (panel 2)analysis of removal of Ebola nucleoprotein (NP) from recombinant viruspreparations (see example 3, experiment 3.3 for details). Lanes A, B, Ccontain product A, B and C, respectively (see FIG. 6 and experiment3.3). For the Western blot analysis, an antibody recognizing NP wasused. The arrows denote NP.

FIG. 8. RP-HPLC analysis of removal of Ebola nucleoprotein (NP) fromrecombinant virus. Products A, B and C were analysed. For details seeexample 3, experiment 3.3. The vertical axis are in AU (×10⁻³). Underthe horizontal axis (elution time), arrow 1 indicates the peak of hexonprotein, arrow 2 indicates peak of NP.

FIG. 9. SDS-PAGE (panel A) and Western blot (panel B) showing theremoval of Ebola nucleoprotein (NP) from recombinant virus preparationsusing high salt and filtration. After anion exchange chromatography thesample was buffer exchanged with a solution comprising 5M NaCl. Thesample was directly filtered through a 0.45 μm Millipac 20 filter(Millipore). Lane 1: before filtration, lane 2: after filtration. Forthe Western blot, an antibody recognizing NP was used. The arrow denotesNP.

FIG. 10. Chromatogram of Ad35 TFF retentate (example 6) loaded on a Q-XLcolumn (panel A) and on a charged filter (panel B). The circle in panelB indicates the extra peak, which is only separated from the virus peakusing the charged filter.

FIG. 11. Disc centrifugation analysis of two fractions of the chargedfilter chromatogram. Panel A shows the sedimentation profile of the Ad35virus peak, panel B shows the sedimentation profile of the extra peak(circled in FIG. 10).

FIG. 12. SDS-PAGE analysis of chromatography fractions Ad35 (see example6). 4-12% bis-tris gel, stained with silver. Gel A shows the fractionsof the charged filter run: 1. marker; 2. start material; 3. flowthrough;4. peak 1 (circled in FIG. 10); 5. Ad35 peak. Gel B shows the fractionsof the Q-XL run: 1. start material; 2. flowthrough; 3. Ad35 peak.

DESCRIPTION OF THE INVENTION

The present invention provides a method for the purification of a virusfrom a host cell, said method comprising the steps of: a) culturing hostcells that are infected with a virus, b) adding nuclease to the cellculture, and c) lysing said host cells to provide a lysate comprisingthe virus. In preferred embodiments, the method further comprises: d)clarification of the lysate. In still more preferred embodiments, themethod further comprises: e) further purifying the adenovirus,preferably with at least one chromatography step. The most importantdifference with the methods hitherto disclosed, is that in those methodsa nuclease is applied only after lysing the cells, or at a later stagein the purification process. According to the present invention, anuclease is added before lysing the cells. As disclosed herein, it hasnow been unexpectedly found that this results in an improvement over theprocesses wherein nuclease is added only after the cells have beenlysed. In the method according to the present invention, the purifiedvirus batch resulting from this process contains less host cell DNA thanwith the method wherein the lysing of cells precedes the nucleaseaddition. In a preferred embodiment, the virus is a recombinantadenovirus. In one embodiment, the nuclease used in step b) isbenzonase®. In one embodiment, the step of lysing the host cells (stepc) is performed with a detergent, which in one embodiment thereof isTriton-X100. In one embodiment, the clarification of the lysate (step d)comprises depth filtration and membrane filtration. In a preferredembodiment thereof, said membrane filtration is performed using acombination of filters having a pore size of 0.8 μm and 0.45 μm, such asa combination filter comprising two asymmetric polyethersulfonemembranes with pore sizes of 0.8 and 0.45 μm, such as a Sartopore™-2combination filter. In one embodiment, the clarified lysate (resultingfrom step d) is subjected to ultrafiltration and/or diafiltration. In apreferred embodiment thereof, the diafiltration results in bufferexchange against a solution comprising 0.8-2.0 M NaCl, or another saltproviding an equivalent ionic strength. In certain preferredembodiments, further purification of the virus (step e) comprises anionexchange chromatography. In another embodiment, said furtherpurification of the virus (step e) comprises a size exclusionchromatography step, preferably in group separation mode. In anotherpreferred embodiment, step e) comprises both anion exchangechromatography and size exclusion chromatography. In certain embodimentsaccording to the invention, the clarified lysate and further purifiedvirus (from step d onwards) are in buffers that are free of detergent,magnesiumchloride and sucrose.

In another aspect, the invention provides a batch of recombinantadenovirus comprising a transgene chosen from the group consisting of:an Ebolavirus nucleoprotein, an Ebolavirus glycoprotein, a Plasmodiumfalciparum circumsporozoite gene, and measles virus hemagglutinin, saidbatch characterized in that it contains less than 0.1 ng host cell DNAper 1E11 viral particles.

The invention further provides a method for the production of a viruscomprising a nucleic acid sequence coding for a nucleic acid bindingprotein, comprising the steps of: a) culturing host cells that have beeninfected with virus, b) subjecting said culture of host cells and saidvirus therein produced to lysis of the host cells to provide a lysatecomprising said virus, c) subjecting the virus to anion exchangechromatography, characterized in that after anion exchangechromatography the virus containing mixture is buffer exchanged with asolution comprising at least 1 M NaCl, or another salt providing anequivalent ionic strength. Preferably, said solution comprises at least1.5 M NaCl, more preferably at least 2 M NaCl, still more preferably atleast 3 M NaCl, still more preferably about 5 M NaCl, or another saltproviding an equivalent ionic strength. Preferably said virus is furtherpurified using filtration through a hydrophilic filter, preferably witha pore size not larger than 1.2 μm, and/or by size exclusionchromatography. The virus preferably is a recombinant virus, morepreferably a recombinant adenovirus. The nucleic acid binding proteinmay be a nuclear protein, such as a nucleoprotein of a haemorrhagicfever virus, such as Ebola, Marburg or Lassa virus, preferably Ebolavirus.

DETAILED DESCRIPTION OF THE INVENTION

Host Cells

A host cell according to the present invention can be any host cellwherein a desired virus can be propagated. For example, the propagationof recombinant adenovirus vectors is done in host cells that complementdeficiencies in the adenovirus. Such host cells preferably have in theirgenome at least an adenovirus E1 sequence, and thereby are capable ofcomplementing recombinant adenoviruses with a deletion in the E1 region.Further the adenovirus may have a deletion in the E3 region, which isdispensable from the Ad genome, and hence such a deletion does not haveto be complemented. Any E1-complementing host cell can be used, such ashuman retina cells immortalized by E1, e.g. 911 (see U.S. Pat. No.5,994,128), E1-transformed amniocytes (See EP patent 1230354),E1-transformed A549 cells (see e.g. WO 98/39411, U.S. Pat. No.5,891,690), GH329:HeLa (Gao et al, 2000, Human Gene Therapy 11:213-219), 293, and the like. Preferably PER.C6™ cells (U.S. Pat. No.5,994,128), or cells derived therefrom are used as host cells, as theyare suitable for the propagation of various different viruses (see e.g.WO 01/38362), including but not limited to recombinant adenoviruses.

Further cell lines and methods for the propagation of recombinantadenoviral vectors have for instance been disclosed in U.S. Pat. No.6,492,169 and in WO 03/104467.

Examples of other useful mammalian cell lines that may be used directlyas host cells for propagating viruses or converted into complementinghost cells for replication deficient virus are Vero and HeLa cells andcell lines of Chinese hamster ovary, W138, BHK, COS-7, HepG2, 3T3, RINand MDCK cells, as known to the person skilled in the art.

Host cells according to the invention are cultured to increase cell andvirus numbers and/or virus titers. Culturing a cell is done to enable itto metabolize, and/or grow and/or divide and/or produce virus ofinterest according to the invention. This can be accomplished by methodsas such well known to persons skilled in the art, and includes but isnot limited to providing nutrients for the cell, for instance in theappropriate culture media. The methods may comprise growth adhering tosurfaces, growth in suspension, or combinations thereof. Culturing canbe done for instance in dishes, roller bottles or in bioreactors, usingbatch, fed-batch, continuous systems, hollow fiber, and the like. Inorder to achieve large scale (continuous) production of virus throughcell culture it is preferred in the art to have cells capable of growingin suspension, and it is preferred to have cells capable of beingcultured in the absence of animal- or human-derived serum or animal- orhuman-derived serum components. Suitable conditions for culturing cellsare known (see e.g. Tissue Culture, Academic Press, Kruse and Paterson,editors (1973), and R. I. Freshney, Culture of animal cells: A manual ofbasic technique, fourth edition (Wiley-Liss Inc., 2000, ISBN0-471-34889-9).

The present invention comprises subjecting cultured host cells that areinfected with virus to lysis. Culturing host cells and infecting themwith a virus is well known to the person skilled in the art. Infectingof host cells can for instance simply be accomplished by exposing thevirus to the appropriate host cell under physiological conditions,permitting uptake of the virus. For certain viruses it is not evennecessary to start with virus per se, as nucleic acid sequences may beused to reconstitute the virus in the cultured cells.

Several aspects of and systems suitable for culturing host cells foradenovirus production can also be found in WO 98/22588, p. 11-28.Methods for culturing cells and propagating viruses in host cells havealso been disclosed in, for example, U.S. Pat. Nos. 6,168,944,5,994,134, 6,342,384, 6,168,941, 5,948,410, 5,840,565, 5,789,390,6,309,650, 6,146,873 and international patent applications WO 01/38362,WO 01/77304 and WO 03/084479.

Viruses

The methods of the instant invention are amenable to a wide range ofviruses, including but not limited to adenoviruses, pox viruses,iridoviruses, herpes viruses, papovaviruses, paramyxoviruses,orthomyxoviruses (such as influenza), retroviruses, adeno-associatedvirus, vaccinia virus, rotaviruses, etc.; adenoviruses beingparticularly preferred. The viruses are preferably recombinant viruses,but can include clinical isolates, attenuated vaccine strains, and soon. In certain embodiments, the present invention is used forconcentrating recombinant viruses, preferably adenoviruses, carrying aheterologous transgene for use in gene therapy or for vaccinationpurposes. For purposes of illustration only, the invention will bedescribed in more detail for recombinant adenovirus, but is in no waylimited thereto.

Adenoviruses

Preferably, the adenoviral vector is deficient in at least one essentialgene function of the E1 region, e.g., the E1a region and/or the E1bregion, of the adenoviral genome that is required for viral replication.In certain embodiments, the vector is deficient in at least oneessential gene function of the E1 region and at least part of thenonessential E3 region (e.g., an Xba I deletion of the E3 region). Theadenoviral vector can be “multiply deficient,” meaning that theadenoviral vector is deficient in one or more essential gene functionsin each of two or more regions of the adenoviral genome. For example,the aforementioned E1-deficient or E1-, E3-deficient adenoviral vectorscan be further deficient in at least one essential gene of the E4 regionand/or at least one essential gene of the E2 region (e.g., the E2Aregion and/or E2B region). Adenoviral vectors deleted of the entire E4region can elicit lower host immune responses. Examples of suitableadenoviral vectors include adenoviral vectors that lack (a) all or partof the E1 region and all or part of the E2 region, (b) all or part ofthe E1 region, all or part of the E2 region, and all or part of the E3region, (c) all or part of the E1 region, all or part of the E2 region,all or part of the E3 region, and all or part of the E4 region, (d) atleast part of the E1a region, at least part of the E1b region, at leastpart of the E2a region, and at least part of the E3 region, (e) at leastpart of the E1 region, at least part of the E3 region, and at least partof the E4 region, and (f) all essential adenoviral gene products (e.g.,adenoviral amplicons comprising ITRs and the packaging signal only). Incase of deletions of essential regions from the adenovirus genome, thefunctions encoded by these regions have to be provided in trans,preferably by the host cell, i.e. when parts or whole of E1, E2 and/orE4 regions are deleted from the adenovirus, these have to be present inthe host cell, for instance integrated in the genome, or in the form ofso-called helper adenovirus or helper plasmids.

The replication-deficient adenoviral vector can be generated by usingany species, strain, subtype, or mixture of species, strains, orsubtypes, of an adenovirus or a chimeric adenovirus as the source ofvector DNA (see for instance WO 96/26281, WO 00/03029), which forinstance may provide the adenoviral vector with the capability ofinfecting certain desired cell types. The adenoviral vector can be anyadenoviral vector capable of growth in a cell, which is in somesignificant part (although not necessarily substantially) derived fromor based upon the genome of an adenovirus. The adenoviral vector maycomprise an adenoviral genome of a wild-type adenovirus of group C,especially of serotype 5 (i.e., Ad5) or Ad2. The adenoviral vector mayalso comprise an adenoviral genome or at least a fiber protein derivedfrom an adenovirus of group B, for instance Ad11, Ad35, Ad51, etc. (seee.g. WO 00/70071), which embodiments have the advantage that lessneutralizing antibodies against these serotypes are encountered in thepopulation, and confer the possibility of targeting other cell types,since the tropism of these adenoviral vectors differs from those derivedfrom Ad5. Of course, the person skilled in the art will know that alsoany other serotype can be applied. The person skilled in the art will beaware of the possibilities to propagate adenoviral vectors of differentserotypes on specific host cells, using methods such as for instancedisclosed in U.S. Pat. No. 6,492,169 or in WO 03/104467, and referencestherein. Adenoviral vectors, methods for construction thereof andmethods for propagating thereof, are well known in the art and aredescribed in, for example, U.S. Pat. Nos. 5,559,099, 5,837,511,5,846,782, 5,851,806, 5,994,106, 5,994,128, 5,965,541, 5,981,225,6,040,174, 6,020,191, and 6,113,913, and Thomas Shenk, “Adenoviridae andtheir Replication”, M. S. Horwitz, “Adenoviruses”, Chapters 67 and 68,respectively, in Virology, B. N. Fields et al., eds., 3d ed., RavenPress, Ltd., New York (1996), and other references mentioned herein. Theconstruction of adenoviral vectors is well understood in the art andinvolves the use of standard molecular biological techniques, such asthose described in, for example, Sambrook et al., Molecular Cloning, aLaboratory Manual, 2d ed., Cold Spring Harbor Press, Cold Spring Harbor,N.Y. (1989), Watson et al., Recombinant DNA, 2d ed., Scientific AmericanBooks (1992), and Ausubel et al., Current Protocols in MolecularBiology, Wiley Interscience Publishers, NY (1995), and other referencesmentioned herein.

Transgenes

In one embodiment, the virus according to the invention is a wild typevirus, or a mutant or part thereof that is still infectious in hostcells according to the invention.

In another embodiment, the virus is a recombinant virus comprisingheterologous information, which may be used in a therapeutic setting forgene therapy purposes, or as an antigen for vaccination purposes. Thisis a preferred embodiment using for instance adenoviral vectors. Theheterologous information is referred to as ‘transgene’. The methodsaccording to the present invention are applicable with a virus,preferably adenovirus, comprising any transgene, and hence the nature ofthe transgene is in itself not material to the present invention.

Several possible transgenes have for instance been described in WO98/22588, p. 42-49. Transgenes that may be present in a virus accordingto the invention may for instance be therapeutic genes, such as tumorsuppressor genes, including but not limited to p53, p16, APC, DCC, NF-1,WT-1, p21, BRCA1, BRCA2, and the like; enzymes, such as cytosinedeaminase, HGPRT, glucocerebrosidase, HSV thymidine kinase or humanthymidine kinase, etc; hormones, such as growth hormone, prolactin,erythropoietin, chorionic gonadotropin, thyroid-stimulating hormone,leptin, ACTH, angiotensin, insulin, glucagon, somatostatin, calcitonin,vasopressin, and the like; interleukins and cytokines, such as IL-1,IL-3, IL-12, G-CSF, GM-CSF, TNF, and the like; replacement genes lackingor mutated in specific disorders, such as ADA, factor IX, CFTR, etc;other therapeutic genes such as angiogenesis inhibitors, cell cycleinhibitors and the like; antisense constructs to inhibit expression offor instance oncogenes, such as ras, myc, jun, bcl, abl, and the like;as well as antigens for vaccines such as viral antigens, for instancederived from a picornavirus, coronavirus, togavirus, flavivirus,rhabdovirus, paramyxovirus, orthomyxovirus, poxvirus, hepadnavirus,reovirus, retrovirus, herpesvirus, and the like, for instance morespecifically antigens from influenza (with as potential antigens forinstance HA and/or NA), hepatitis B (with as potential antigen hepatitisB surface antigen), West Nile Virus, rabies, SARS-CoV, herpes simplexvirus 1 and 2, measles, small pox, polio, HIV (with antigens e.g. HIV-1derived gag, env, nef, or modifications thereof including codonoptimized versions, see for instance WO 02/22080), Ebola, Marburg, Lassavirus; or bacterial antigens, fungal antigens, parasitic (includingtrypanosomes, tapeworms, roundworms, helminths, malaria, etc) antigens,and the like. Clearly, the person skilled in the art will choose thegene of interest that is useful in the envisaged therapeutic setting, beit in gene therapy and/or in vaccination, and is not confined to thelist above. It is also clear that control regions for the transgene arepreferably present in recombinant viral vectors aimed at expression ofthe transgene, for instance including a promoter and a polyadenylationsignal. These are all aspects well known to the person skilled in theart, and need not further be elaborated here. Several control regionsare discussed in WO 98/22588, p. 49-55.

Some adenoviruses used in the present invention are further discussed inthe examples.

Lysing Host Cells

After infection of an adenovirus, the virus replicates inside the celland is thereby amplified. Adenovirus infection results finally in thelysis of the cells being infected. The lytic characteristics ofadenovirus therefore permits two different modes of virus production.The first mode is harvesting virus prior to cell lysis, employingexternal factors to lyse the cells. The second mode is harvesting virussupernatant after (almost) complete cell lysis by the produced virus(see e.g. U.S. Pat. No. 6,485,958, describing the harvesting ofadenovirus without lysis of the host cells by an external factor). Forthe latter mode, longer incubation times are required in order toachieve complete cell lysis, and hence high yields of virus.Furthermore, the gradual spill of the host cell contents into the mediummay be detrimental to the integrity and yield of the obtained viruses.Hence, it is preferred to employ external factors to actively lyse thecells, according to the invention.

Methods that can be used for active cell lysis are known to the personskilled in the art, and have for instance been discussed in WO 98/22588,p. 28-35. Useful methods in this respect are for example, freeze-thaw,solid shear, hypertonic and/or hypotonic lysis, liquid shear,sonication, high pressure extrusion, detergent lysis, combinations ofthe above, and the like. In one embodiment of the invention, the cellsare lysed using at least one detergent. Use of a detergent for lysis hasthe advantage that it is an easy method, and that it is easily scalable.In another embodiment, the cells are lysed by shear using hollow fiberultrafiltration, such as described in WO 03/084479.

Detergents

Detergents that can be used according to the present invention, and theway they are employed, are generally known to the person skilled in theart. Several examples are for instance discussed in WO 98/22588, p.29-33.

Detergents, as used herein, can include anionic, cationic, zwitterionic,and nonionic detergents. Exemplary detergents include but are notlimited to taurocholate, deoxycholate, taurodeoxycholate, cetylpyridium,benzalkonium chloride, ZWITTERGENT-3-14®, CHAPS(3-[3-Cholamidopropyl)dimethylammoniol]-1-propanesulfonate hydrate,Aldrich), Big CHAP, Deoxy Big CHAP, Triton X-100®, Triton X-114®, C12E8,Octyl-B-D-Glucopyranoside, PLURONIC-F68®, TWEEN-20®, TWEEN-80®(CALBIOCHEM® Biochemicals), Thesit®, NP-40®, Brij-58®, octyl glucoside,and the like. It is clear to the person skilled in the art that theconcentration of the detergent may be varied, for instance within therange of about 0.1%-5% (w/w). In certain embodiments the detergent ispresent in the lysis solution at a concentration of about 1% (w/w). Insome pilot experiments of the inventors, use of Triton resulted in lessviscous solutions than some other detergents tested (Tween 20, Tween 80,deoxycholate). In one embodiment of the present invention, the detergentused is Triton X-100.

Nuclease

The present invention employs nuclease to remove contaminating, i.e.mostly host cell, nucleic acids. Exemplary nucleases suitable for use inthe present invention include Benzonase®, Pulmozyme®, or any other DNaseand/or RNase commonly used withing the art. In preferred embodiments ofthe invention, the nuclease is Benzonase®, which rapidly hydrolyzesnucleic acids by hydrolyzing internal phosphodiester bonds betweenspecific nucleotides, thereby reducing the viscosity of the cell lysate.Benzonase® can be commercially obtained from Merck KGaA (code W214950).

The concentration in which the nuclease is employed is preferably withinthe range of 1-100 units/ml.

According to the invention, the nuclease is employed before the cellsare lysed. It may be added just seconds prior to (or virtuallyconcomitant with) the lysis step, but preferably the nuclease is addedto the culture at least one minute before the lysis step. The cellculture with the added nuclease can then be incubated above processtemperature, e.g. around 40° C., or at the culturing temperature (e.g.between about 35° C. to about 37° C.), or at room temperature (around20° C.) or lower (e.g. around 0° C.), wherein in general longerincubation times are required at lower temperature to achieve the sameresult (see Benzonase® brochure Merck KGaA code W 214950). As anon-limiting example, the incubation can for instance be performed atabout 37° C., for about 10 minutes, after which the cells are lysed.Obviously, the nuclease can and preferably will still actively degradenucleic acid after the lysis step, and in certain embodiments accordingto the present invention the incubation of the cells with endonucleaseafter lysis is prolonged for about 50 minutes (resulting in a total timeof the nuclease treatment of about 1 hour, although this time mayeffectively be still longer, because it is anticipated that the nucleasewill still be functional until it is removed in subsequent purificationsteps). This is considerably shorter than the overnight incubationdisclosed in WO 98/22588. Of course, longer incubation, such as forinstance 2 hours or overnight or longer incubation (in Benzonase®brochure Merck KGaA code W 214950, data for up to 30 hours incubationare provided) is also possible according to the methods of the presentinvention, but is not required to obtain acceptable results. It will beclear that the ‘lysis step’ (i.e. subjecting the cells containing thevirus produced therein to lysis) as used in these embodiments, is meantto be a lysis step employing external factors (see under ‘lysing hostcells’ above), such as a detergent. Obviously, during the culturing ofthe cells wherein the adenovirus is propagated, some cells may alreadylyse because of the virus in absence of any external lysis factors.Hence, in preferred embodiments, such lysis in the absence of externalfactors has occurred in less than 50%, preferably less than 40%, morepreferably less than 30%, still more preferably less than 20% of thehost cells, when nuclease treatment is started, i.e. preferably nucleaseis added when the cells have a viability of at least 50%, 60%, 70%, 80%,respectively.

Although not preferred (see above), methods that are dependent on lysisof the host cells in the absence of external factors can be used.Processes involving ‘spontaneous’ lysis have been described, wherein theuse of Benzonase is discouraged (see U.S. Pat. No. 6,485,958). However,according to the present inventors it will be beneficial also in suchsystems to add nuclease during the later stages of the culture, i.e.preferably when the host cells wherein the virus is propagated stillhave a viability of at least 5%, more preferably at least 10%, stillmore preferably at least 20% (i.e. when less than 95%, 90%, 80% of thecells are lysed, respectively). It is anticipated that this will improvethe process in quality of the obtained virus when this step would beemployed. It is therefore another aspect of the invention to provide amethod for the purification of a virus that is capable of lysing hostcells from host cells, said method comprising the steps of: a) culturinghost cells comprising a virus capable of lysing said host cells, b)harvesting virus following their release into culture fluid withoutlysis of the host cells by an external factor, characterized in that anuclease is added to the culture before 95% of the host cells has beenlysed. In certain embodiments, the nuclease is added to the culturebefore 90%, preferably 80% of the host cells has been lysed. The findingof the optimal moment (i.e. corresponding to the optimal percentage ofcells that has been lysed) to add the nuclease in these aspects of theinvention will depend on the amount of nuclease added and the decreasein specific activity of the nuclease during incubation, and can beempirically found by the person skilled in the art, now the advantage ofthe addition of nuclease to the culture per se has been disclosed by thepresent inventors. Clearly, the obtained lysate according to this aspectof the invention can be further purified employing methods and steps asdiscussed herein, such as filtration and chromatography.

International patent application WO 03/097797 describes alternativemethods for purifying adenovirus particles from cell lysates, comprisingthe addition of a selective precipitation agent to precipitate impurityDNA. Although it is stated therein that a nuclease step is not requiredwhen that method is used, such a step in a later stage of the procedureis used for robustness. The method according to the present invention,including the step of adding a nuclease prior to host cell lysis, mightsuitably be combined with the addition of a selectively precipitationagent after lysis, thereby making a step of nuclease addition later inthe process (as preferred in WO 03/097797) potentially superfluous.

International patent application WO 02/070673 employs a continuouscentrifugation method for isolation of virus from host cells: the cellculture is subjected to continuous centrifugation under conditionseffective to concentrate the cells into a pellet, and the pelleted cellsare ejected from the centrifuge into a collection receptacle underconditions effective to lyse the cells and thereby obtain a lysate.Clearly, lysing the cells according to that method is also within thescope of ‘lysing the host cells’ according to the present invention, andhence it is anticipated that also such a method should benefit from thepresent invention, i.e. addition of nuclease to the cell culture beforesubjecting it to the continuous centrifugation method, the thus improvedmethod resulting in lower nucleic acid contamination in the lysate andhence in the final purified product.

Clarification

In preferred embodiments of the invention, the host cell lysatecomprising the virus is clarified. Clarification may be done by afiltration step, removing cell debris and other impurities. Suitablefilters may utilize cellulose filters, regenerated cellulose fibers,cellulose fibers combined with inorganic filter aids (e.g. diatomaceousearth, perlite, fumed silica), cellulose filters combined with inorganicfilter aids and organic resins, or any combination thereof, andpolymeric filters (examples include but are not limited to nylon,polypropylene, polyethersulfone) to achieve effective removal andacceptable recoveries. In general, a multiple stage process ispreferable but not required. An exemplary two or three-stage processwould consist of a course filter(s) to remove large precipitate and celldebris followed by polishing second stage filter(s) with nominal poresizes greater than 0.2 micron but less than 1 micron. The optimalcombination may be a function of the precipitate size distribution aswell as other variables. In addition, single stage operations employinga relatively tight filter or centrifugation may also be used forclarification. More generally, any clarification approach includingdead-end filtration, microfiltration, centrifugation, or body feed offilter aids (e.g. diatomaceous earth) in combination with dead-end ordepth filtration, which provides a filtrate of suitable clarity to notfoul the membrane and/or resins in the subsequent steps, will beacceptable to use in the clarification step of the present invention.

In one embodiment, depth filtration and membrane filtration is used.Commercially available products useful in this regard are for instancementioned in WO 03/097797, p. 20-21. Membranes that can be used may becomposed of different materials, may differ in pore size, and may beused in combinations. They can be commercially obtained from severalvendors.

It has now been found by the present inventors that certain membranesunexpectedly give superior results in the process of the invention,providing much improved clarification compared to other membranes (seeexample 4).

It is therefore a preferred embodiment of the invention to use acombination of 0.8 μm and 0.45 μm filters, preferably Sartopore-2filters, for clarification.

Ultra filtration/Diafiltration

In certain embodiments of the invention, the virus suspension issubjected to ultrafiltration/diafiltration at least once during theprocess, e.g. for concentrating the virus and/or buffer exchange, and/orfor concentration and diafiltration of the clarified harvest. Theprocess used to concentrate the virus according to the method of thepresent invention can include any filtration process (e.g.,ultrafiltration (UF)) where the concentration of virus is increased byforcing diluent to be passed through a filter in such a manner that thediluent is removed from the virus preparation whereas the virus isunable to pass through the filter and thereby remains, in concentratedform, in the virus preparation. UF is described in detail in, e.g.,Microfiltration and Ultrafiltration: Principles and Applications, L.Zeman and A. Zydney (Marcel Dekker, Inc., New York, N.Y., 1996). Apreferred filtration process is Tangential Flow Filtration (“TFF”) asdescribed in, e.g., MILLIPORE catalogue entitled “Pharmaceutical ProcessFiltration Catalogue” pp. 177-202 (Bedford, Mass., 1995/96). TFF iswidely used in the bioprocessing industry for cell harvesting,clarification, and concentration of products including viruses. Thesystem is composed of three distinct process streams: the feed solution,the permeate and the retentate. Depending on application, filters withdifferent pore sizes may be used. In one embodiment of the presentinvention the retentate is the product, and can be used for furtherpurification steps if desired. For this embodiment, the particularultrafiltration membrane selected will have a pore size sufficientlysmall to retain virus but large enough to effectively clear impurities.Depending on the manufacturer and membrane type, for adenovirus nominalmolecular weight cutoffs (NMWC) between 100 and 1000 kDa may beappropriate, for instance membranes with 300 kDa or 500 kDa NMWC. Themembrane composition may be, but is not limited to, regeneratedcellulose, polyethersulfone, polysulfone, or derivatives thereof. Themembranes can be flat sheets or hollow fibers. UF is generally referredto filtration using filters with a pore size of smaller than 0.1 μm.Products are generally retained, while volume is reduced throughpermeation. The two most widely used geometries for TFF in thebiopharmaceutical industry are plate & frame and hollow fiber modules.Hollow fiber units for ultrafiltration and microfiltration weredeveloped by Amicon and Ramicon in the early 1970s (Cheryan, M.Ultrafiltration Handbook), even though now there are multiple vendorsincluding Spectrum and A/G Technology. The hollow fiber modules consistof an array of self-supporting fibers with a dense skin layer that givethe membranes its permselectivity. Fiber diameters range from 0.5 mm-3mm. An advantage of hollow fiber modules is the availability of filtersfrom small membrane areas (ca. 16 cm²) to very large membrane areas (ca.28 m²) allowing linear and simple scale-up. In certain preferredembodiments according to the invention, hollow fibers are used for TFF.These are reported to give less shear and a better viralparticle/infectious unit (VP/IU) ratio than flat screen membranes. Incertain embodiments, hollow fibers of 0.05 μm are used according to theinvention.

Diafiltration (DF), or buffer exchange, using ultrafilters is an idealway for removal and exchange of salts, sugars, non-aqueous solventsseparation of free from bound species, removal of material of lowmolecular weight, or rapid change of ionic and/or pH environments.Microsolutes are removed most efficiently by adding solvent to thesolution being ultrafiltered at a rate equal to the UF rate. This washesmicrospecies from the solution at a constant volume, purifying theretained virus. The present invention utilizes a DF step to exchange thebuffer of the lysate prior to further chromatography or otherpurification steps. According to one embodiment of the invention DF byTFF is performed for buffer exchange, wherein the addition of bufferequals the removal of permeate.

UF/DF can be used to concentrate and/or buffer exchange the virussuspensions according to the present invention in different stadia ofthe purification process, e.g. the lysate and/or further purified virussuspensions such as those that have undergone chromatography.

In one embodiment according to the invention, the lysate is concentratedby UF/DF 5-fold, and the resulting concentrated virus suspension isbuffer exchanged with 6 diafiltration volumes (DFV) of a buffercomprising 1 M NaCl, using a constant volume diafiltration method. Itwas found that this high salt concentration significantly improves thequality of the resulting virus, as many undesired proteins were lostduring this step (see example 2). It is therefore a preferred embodimentaccording to the invention that the clarified lysate is exchangedagainst a solution comprising 0.8-2.0 M NaCl, e.g. around 1 M NaCl, oranother salt providing an equivalent ionic strength. It will be clear tothe person skilled in the art that both the anion and the cation of thesalt can be changed.

Before the virus suspension is subjected to anion exchangechromatography, it may be buffer exchanged with a buffer comprising 0.4M NaCl, or another salt providing an equivalent ionic strength. In oneembodiment, this is accomplished by constant volume diafiltration, using4 DFVs of the desired buffer.

Further Purification

According to preferred embodiments of the present invention, the virussuspension that has been obtained by the method according to the presentinvention, preferably after clarification of the lysate, is furtherpurified, e.g. by methods generally known to the person skilled in theart. This may for instance be achieved by density gradientcentrifugation, as for instance discussed in WO 98/22588, p. 59-61.

Preferably however, further purification employs at least onechromatography step, as for instance discussed in WO 98/22588, p. 61-70.Many processes have been described for the further purification ofviruses, wherein chromatography steps are included in the process. Theperson skilled in the art will be aware of these processes, and can varythe exact way of employing chromatographic steps to optimize the processof the invention.

It is for instance possible to purify certain viruses by a combinationof anion exchange and cation exchange chromatography steps, see U.S.Pat. No. 6,008,036.

It is also possible to employ a hydroxyapatite medium for purifyingadenovirus, see WO 02/44348.

A reversed-phase adsorption step might also be used, as for instancedescribed in WO 03/097797, p. 26.

For adenovirus purification, it is preferred to use at least one anionexchange chromatography step. After the anion exchange chromatographystep, the virus may be sufficiently pure. In certain embodiments howevera size exclusion chromatography step is further performed to increasethe robustness of the process. This step may be prior to or after theanion exchange chromatography step. Obviously, other purification stepsmay also be suitably combined with an anion exchange chromatographystep.

The use of anion exchange chromatography for adenovirus purification hasbeen extensively described, and this aspect is therefore well within thereach of the person skilled in the art. Many different chromatographymatrices have been employed for purification of adenovirus and aresuitable, and the person skilled in the art can easily find the optimalanion exchange material for purifying the virus, for instance guided bythe following art.

U.S. Pat. No. 5,837,520 (see also Huyghe et al., 1995, Human GeneTherapy 6: 1403-1416) describes a method of purifying adenovirus whereinthe host cell lysate is treated with a nuclease, followed by anionexchange and metal ion affinity chromatography.

U.S. Pat. No. 6,485,958 describes the use of strong anion exchangechromatography for purification of recombinant adenovirus. Anionexchange chromatography has been employed with fluidized bed columns forthe purification of adenovirus particles, see WO 00/50573.

Further, expanded bed anion exchange chromatography, and certainchromatographic resins for anion exchange chromatography forpurification of adenovirus particles have been described in U.S. Pat.No. 6,586,226.

In addition to anion exchange columns, anion exchange membranechromatography products such as those produced by Pall (e.g. Mustang™series) and Sartorius (e.g. Sartobind series) are suitable. For use ofthese filters and their advantages in adenovirus purification see forinstance WO 03/078592. Clearly, employment of such filters also fallswithin the scope of the term ‘anion exchange chromatography’ as usedherein.

U.S. Pat. No. 6,537,793 describes the purification of adenoviralparticles from host cells using ion-exchange chromatography, inparticular teaching a preference for Q Sepharose XL types ofchromatographic support for this purpose. In one embodiment of thepresent invention, an adenovirus is further purified using a Q SepharoseXL column.

As described above, the process may further suitably employ a sizeexclusion chromatography step.

International application WO 97/08298 describes the purification ofadenoviruses using certain chromatographic matrices to prevent damage tothe viruses, including anion exchange and size exclusion steps.

U.S. Pat. No. 6,261,823 describes a method for purifying adenoviruswherein the adenovirus preparation is subjected to anion exchangechromatography followed by size exclusion chromatography. In the sizeexclusion step, a group separation of viral particles from impurities oflow molecular weight is achieved. According to certain embodiments ofthe present invention, about 15-30%, preferably about 20% of the columnvolume is loaded on the size exclusion column (group separation mode ofsize exclusion chromatography).

Hence, in a preferred embodiment of the invention, an adenovirussuspension that has been prepared according to the method of theinvention is further purified using an anion exchange chromatographystep and a size exclusion chromatography step.

WO 03/078592 describes the use of high throughput anion exchange filters(i.e. a charged filter that contains anion exchange groups) foradenovirus (Ad5) purification. The following advantages are describedfor such charged filters compared to anion exchange columns: (i) fasterflow rates, (ii) higher binding capacity, (iii) higher virus recovery,(iv) no packing or cleaning validation required for clinical use, and(v) no lifetime issues or storage issues when disposable filtercartridges are used. As described above, the use of such anion exchangefilters is an embodiment of the present invention, and is an embodimentconsidered included within the scope of ‘anion exchange chromatography’in the present invention. However, in addition to being an equivalentfor column chromatography, the present inventors have surprisingly foundan advantage for purifying adenovirus serotype 35 (Ad35) using an anionexchange filter, over the use of an anion exchange column: certainadenovirus proteins that were not incorporated into adenovirus particlesare separated from the adenovirus particles by use of an anion exchangefilter, not by an anion exchange column. Such free adenovirus proteinswere not previously found in preparations of recombinant adenovirusparticles and would normally go undetected, but now can be removed usingthe step of subjecting a recombinant adenovirus preparation comprisingfree adenovirus proteins to a charged filter that contains anionexchange groups. This effect of the use of the charged filter was notnoted in WO 03/078592. In addition, WO 03/078592 does not disclose theemployment of anion exchange filters for the purification of Ad35, orother adenovirus particles of subgroup B. The invention thereforeprovides a method for removing free adenovirus proteins from arecombinant adenovirus preparation, comprising the step of: subjecting arecombinant adenovirus preparation comprising free adenovirus proteinsto a charged filter that contains anion exchange groups. Without wishingto be bound by theory, it is conceivable that the possibly somewhatlower stability of recombinant adenovirus particles of subgroup B (seee.g. WO 2004/001032) gives rise to the hitherto undetected freeadenovirus proteins that appear not incorporated into adenovirusparticles. Hence, this particular method according to the invention maybe particularly beneficial for purification of recombinant adenovirus ofsubgroup B, such as Ad35, Ad11, etc. However, it is also possible thatthe method improves purification of the more stable Ad5 or Ad2 basedadenovirus. The invention provides the use of an anion exchange filterfor the removal of free (i.e. not incorporated into viral particles)adenovirus proteins from a recombinant adenovirus preparation.Preferably, said recombinant adenovirus preparation comprisesrecombinant subgroup B adenovirus, such as recombinant Ad35. Theinvention also provides a method for purification of recombinantsubgroup B adenovirus particles, such as Ad35 particles, the methodcomprising a step of subjecting the recombinant subgroup B, such asAd35, particles to an anion exchange filter purification step. Anionexchange filters suitable for use in these methods of the invention areknown in the art and commercially available (see WO 03/078592,paragraphs [40]-[41]), e.g. from Pall (e.g. Mustang™ series) and fromSartorius (e.g. Sartobind series).

Buffers

Many buffers can be used during purification of the virus according tothe present invention. In several embodiments of the present invention,buffers used for UF/DF and anion exchange chromatography in generalcontained 0.4-1.0 M NaCl/50 mM TRIS pH 7.5, wherein the concentrationsof NaCl were dependent on the process step. In certain preferredembodiments, the buffers used after clarification are free of detergent,magnesium chloride and sucrose. The absence of these additivesdistinguishes these buffers from those used in known establishedprotocols. Nevertheless, when the methods according to the presentinvention are employed, a purified and substantially non-aggregatedadenovirus is obtained. An advantage of the use of buffers without theseadditives is that they are easier to prepare, cheaper, and that there isno need to test for removal of the additives.

In one embodiment according to the invention, the adenovirus is bufferexchanged during group separation to—and finally stored in—the bufferthat is also used for the Adenovirus World Standard (Hoganson et al,Development of a stable adenoviral vector formulation, BioprocessingMarch 2002, p. 43-48): 20 mM Tris pH 8, 25 mM NaCl, 2.5% glycerol.

Obviously, many other buffers can be used, and several examples ofsuitable formulations for the storage and pharmaceutical administrationof purified (adeno) virus preparations can for instance be found inEuropean patent no. 0853660, and in international patent applications WO99/41416, WO 99/12568, WO 00/29024, WO 01/66137, WO 03/049763.

Vectors with Specific Inserts

In the art, the transgene itself is generally regarded as irrelevant forthe purification process. However, as shown herein, the transgene may inspecific cases by its expression in the host cell or in the virusinfluence properties of the virus or may have an influence on theprocess of purifying the virus.

One such, non-limiting, specific case as found by the present inventors,is where the transgene is the Ebolavirus nucleoprotein. Purifying anadenoviral vector containing the Ebolavirus nucleoprotein gene with thestandard purification procedure results in co-purifying the expressedEbolavirus nucleoprotein. No co-purification of several other transgeneexpressed proteins was observed (for instance not with Ebolaglycoprotein dTM (Sudan), Ebola Glycoprotein dTM (Zaire), measleshemagglutinin protein (MV-H)). This suggests a specific interactionbetween the Ebola nucleoprotein and Adenovirus, which seems to depend onthe characteristics of the Ebola nucleoprotein. Other nucleic acidbinding proteins are expected to have similar characteristics and areexpected to have an interaction with Adenovirus resulting inco-purification as well. For adenoviruses having such transgenes,including nucleic acid binding proteins, such as nucleoproteins, such asEbolavirus nucleoprotein, it is beneficial to exchange the buffer tosalt concentrations that are even higher than 1 M NaCl, and use forinstance 2-5 M NaCl buffers to improve the final product quality (seeexample 3). Buffer exchange may suitably be performed by TFF.

Alternatively, other methods for buffer exchange could be used, forinstance the salt could be added to the virus suspension directly in agradual way by addition of the solid material or concentration solution.This aspect of the invention may be beneficially combined with otheraspects of the invention, for instance with adding the nuclease beforelysis, but is not limited thereto. It is described herein that use ofsuch high salt buffers unexpectedly does not result in aggregationproblems, nor in significant deterioration of the infectivity orintegrity of the purified viral particles. In this aspect of theinvention, the buffer exchange step preferably takes place after theelution of the virus from anion exchange chromatography, and preferablybefore a further purification step. Such a further purification step mayfor instance be a size exclusion step in group separation mode. Thislast step can be used for polishing the virus suspension, i.e. removingminor impurities that may still be present after anion exchange, butalso for buffer exchange directly on the group separation column.Alternatively, instead of size exclusion, the further purification stepmay comprise a filtration of the virus suspension comprising the highsalt concentration through a hydrophilic filter, such as a Durapore PVDFfilter (e.g. Millipac from Millipore) or a Sartopore 2 filter. Thefilter preferably has a pore size of 1.2 μm, more preferably smaller,e.g. 1.0 μm, still more preferably smaller, e.g. 0.8 μm, 0.45 μm or 0.22μm. Unexpectedly, the nucleoprotein (NP) of Ebolavirus was found to beseparated from a recombinant adenovirus under these conditions by beingretained by the filter, while NP—having a molecular weight of about 100kD—was expected to pass through the filter pores together with theadenovirus. Use of these filters provides a fast solution for separatingthe nucleoprotein from the virus, as no prolonged incubation in highsalt is required for this procedure, while it allowed complete removalof the nucleoprotein from the virus (FIG. 9). Of course, a sizeexclusion chromatography step may still be employed after such afiltration step, to remove other minor contaminants and/or for bufferexchange.

Use of high salt for removing DNA binding proteins is an aspect of theinvention that is expected to be useful for other viruses thanadenoviruses as well. Possibly another column chromatography step may inthat case be applied instead of anion exchange chromatography. Theimportant factor seems to be the removal of sufficient contaminatingmaterial before the high salt step is applied, and of course thisremoval could be achieved by other means than anion exchangechromatography, also for recombinant adenoviruses.

Hence the invention further provides a method for the production of avirus comprising a nucleic acid sequence coding for a nucleic acidbinding protein, comprising the steps of: a) culturing host cells thathave been infected with virus, b) subjecting said culture of host cellsand said virus therein produced to lysis of the host cells to provide alysate comprising said virus, c) subjecting the virus to anion exchangechromatography, characterized in that after anion exchangechromatography the virus containing mixture is buffer exchanged with asolution comprising at least 1 M NaCl, or another salt providing anequivalent ionic strength.

Preferably, the virus is further purified using at least one stepcomprising either filtration though a hydrophilic filter, and/or usingat least one step comprising size exclusion chromatography. For theseembodiments, a solution comprising at least 1 M NaCl or another saltproviding equivalent ionic strength is referred to as a ‘high salt’solution. Clearly, both the anion and the cation can be varied as isknown to the person skilled in the art, as long as sufficient ionicstrength is provided without precipitation or other undesiredside-effects such as inactivation of the virus, as the method likelydepends on the breaking of ionic interactions between the DNA bindingprotein and the purified virus. For example, NaCl may be in part orwholly substituted for other salts, such as for instance KCl, sodiumphosphate, CsCl, LiCl, (NH₄)₂SO₄, NH₄Cl, NaBr, NaI, KBr, KI, KNO₃,NaHCO₃, KHSO₄, etc. A 5× dilution of the buffer used in the example ofthe invention (comprising 5 M NaCl) had a conductivity of 78-79 mS/cm.Buffers containing other salts, and having a similar or higherconductivity can for instance now easily be tested for suitability inremoving DNA binding proteins from partially purified virus, accordingto the invention. It is expected that this embodiment will work up tosaturation of the NaCl concentration (this is about 6 M NaCl), but forpractical reasons it is preferred to use buffers that are not saturated,e.g. 5 M NaCl. Preferably, the solution comprises at least 1.5 M NaCl,or another salt providing an equivalent ionic strength. More preferablythe solution comprises at least 2 M NaCl, or another salt providing anequivalent ionic strength. More preferably, the solution comprises atleast 3 M NaCl, or another salt providing an equivalent ionic strength.More preferably, the solution comprises at least 4 M NaCl, or anothersalt providing an equivalent ionic strength. Even more preferably, thesolution comprises around 5 M NaCl, or another salt providing anequivalent ionic strength. The high salt solution comprising the virusmay be incubated for a certain time, preferably at least one hour, morepreferably at least two hours. In general, the examples show anincreased purification of the DNA binding protein from the virus whenincubation is longer, at least up to overnight. Further, a higher ionicstrength appears to improve the purification. Hence, it is conceivablethat even at ionic strengths of 1 M or 1.5 M NaCl and prolongedincubation, e.g. for at least two days, or one week, there may bepurification of the DNA binding protein from the virus. This can beroutinely checked by the experiments described herein. Overnightincubation of recombinant adenovirus expressing Ebolavirus nucleoproteinin a buffer comprising 5 M NaCl, removed the contaminating nucleoproteinfrom the virus to below detection limits, and is therefore a preferredembodiment of the invention. In preferred embodiments, the virus is arecombinant adenovirus. In certain embodiments, said nucleic acidbinding protein is a nucleoprotein of a virus. In certain embodimentsthereof the nucleic acid binding protein is the Ebolavirusnucleoprotein. In preferred embodiments, the buffer exchange step takesplace after anion exchange chromatography and before a filtration and/orsize exclusion chromatography step. It is further preferred to include anuclease treatment of the lysate, whereby preferably the nuclease isadded to the cell culture before lysis is complete, in accordance withother aspects of the invention. Instead of high salt or in additionthereto, detergent may be added to purify the virus from contaminatingDNA binding protein. In one experiment, the inventors have shown thataddition of 1% Tween 20 also significantly reduced the contaminatingnucleoprotein from recombinant adenovirus expressing Ebolanucleoprotein. Of course, other detergents can suitably be tested, andthe concentration may be varied, e.g. between bout 0.2% and 5%, to findoptimal conditions for removal of DNA binding proteins from recombinantvirus preparations according to the invention. In this aspect,preferably at least 1% detergent is added. The first experiments of theinventors however have indicated a higher reproducibility of high saltincubation for this purpose, and therefore this is preferred.

Batches of Recombinant Adenovirus

In one aspect, the invention provides a batch of recombinant adenoviruscomprising a transgene chosen from the group consisting of: anEbolavirus nucleoprotein, an Ebolavirus glycoprotein, a Plasmodiumfalciparum circumsporozoite gene, measles virus hemagglutinin, saidbatch characterized in that it contains less than 0.1 ng host cell DNAper 1E11 viral particles. Of course, these transgenes optionally maycontain deletions, additions, and/or mutations compared to the wild-typesequences found in nature, including all isolates or subtypes, withoutdeviating from the scope of this aspect of the invention. Clearly, foradministration to subjects it is advantageous, if not already requiredfor regulatory purposes, to have batches with such low amounts ofcontaminating host cell DNA available. In preferred aspects, the batchis characterized in that it contains less than 0.08 ng, more preferablyless than 0.06 ng, still more preferably less than 0.04 ng host cell DNAper 10¹¹ viral particles.

EXAMPLES

The following examples are included to further illustrate the inventionby means of certain embodiments of the invention, and are not to beconstrued to limit the scope of the present invention in any way.

Example 1 Addition of Nuclease to the Cell Culture Instead of to theHost Cell Lysate Improves the Process for Virus Purification

In this example it is shown that addition of nuclease to the cellculture before lysing the cells reduces the amount of residual host cellDNA in the final purified bulk.

In runs 1 and 2a 10 liter PERC.6® cell culture was lysed with 1% TritonX-100® (Sigma) at day 2.5 after infection with an adenoviral vector.Thirty minutes after lysis, Benzonase® (Merck KgaA, 50 units/ml) andMgCl₂ (2 mM) were added. After another 30 minutes the TritonX-100®/Benzonase® (T/B) harvest was clarified by filtration. Thistherefore was a run according to processes known in the art.

In runs 3-8, Benzonase® (50 U/ml) and MgCl₂ (2 mM) were added to 10liter PERC.6 cell culture (day 2.5 post infection), and after 10 minutesincubation the cells were lysed with 1% Triton X-100®. After anadditional incubation of 50 minutes the Benzonase®/Triton X-100® (B/T)harvest was clarified by filtration.

The difference with the processes known from the art therefore is in theorder in which the nuclease (Benzonase®) and the detergent (TritonX-100®) were added: classically first the cells are lysed, andsubsequently nuclease is added (referred to herein as T/B harvest),whereas in the process according to the invention, first nuclease isadded and subsequently the cells are lysed (referred to herein as B/Tharvest). This is schematically shown in FIG. 1.

The samples were then further purified. Clarification was performed bydepth filtration (0.5 μm Clarigard filter, Millipore) followed byfurther clarification over a 0.8/0.45 μm Sartopore 2 (Sartorius) filter.The clarified material was concentrated 5 times over a 0.05 μm hollowfiber (Spectrum), followed by diafiltration with subsequently 6 volumesof 1.0 M NaCl/50 mM TRIS pH 7.5 and 4 volumes of 0.4 M NaCl/50 mM TrispH 7.5. The diafiltered retentate was loaded onto a Sepharose Q-XL(Amersham) column and the virus fraction was eluted with 0.55 M NaCl/50mM TRIS pH 7.5. This fraction was further purified and buffer exchangedwith a Sepharose 4 FF (Amersham) column. The generated purified bulk wasconcentrated to the desired concentration with a hollow fiber (0.05 μmporesize, Spectrum), 0.22 μm filtered and aliquotted. Purified bulksamples were analysed for residual host cell DNA by Q-PCR.

The T/B treatment resulted in a reduction of DNA that after furtherdownstream processing could just meet the required specification in thefilled and finished material. Regulatory requirements for residual hostcell DNA for life virus formulations are <10 ng per dose (assumed that adose contains 1E11 viral particles).

As is shown in Table 1, reversing the Triton X-100® and Benzonase® stepsreduced the amount of residual host cell DNA in the purified bulksignificantly: by the addition of nuclease before active cell lysis theamount of residual host cell DNA could be reduced 10 to 40 times, toless than 0.1 ng/1E11 viral particles.

Further, it is clear from SDS-PAGE analysis (FIG. 2) that uponclarification by depth and membrane filtration of a B/T harvest a numberof host cell proteins, among which a significant amount of histonproteins (M_(w) around 10-20 kD on gels, identity confirmed by massspectrometry), was removed during clarification while these proteins areclearly still present in the clarified T/B harvest.

Hence, the process according to the invention results in significantadvantages over those known from the prior art. Without wishing to bebound by theory, possible explanations for the differences between runs1 and 2 (T/B) on one side and runs 3-8 (B/T) on the other side mayinclude:

1. Upon addition of Benzonase® the DNA released from cells lysed due tovirus production can already be digested. As soon as DNA is releasedfrom cells lysed by Triton, the Benzonase® is present to immediatelydigest the DNA, thereby preventing the formation of large DNAaggregates. Digestion of non-aggregated DNA is probably more effectivethan digestion of major DNA aggregates.

2. The total incubation time of Benzonase® increases with 30 minutes,resulting in more effective digestion (see Benzonase® brochure MerckKGaA code W 214950).

3. Possibly larger histon complexes are formed when the DNA is digestedimmediately upon release and these larger particles are retained by theclarification filters. Retainment of histon-DNA complexes duringclarification might also have contributed to reduction of residual hostcell DNA.

Several anion exchange resins have been tested e.g. QAE 550C and Super Q650M (purchased from Tosoh), Q Sepharose HP, ANX Sepharose 4FF, DEAESepharose, Q Sepharose XL, Q Sepharose Big Bead and Q Sepharose FF(purchased from Amersham). Although all these resins were suitable forthe purification of the recombinant adenoviruses, we found that QSepharose XL was best suitable for our purpose based on separation ofvirus from host cell proteins and host cell DNA, and flowcharacteristics.

Several size exclusion resins were tested e.g. Sephacryl S300, SephacrylS500 Sepharose 4FF and Sepharose 6 FF (all purchased from Amersham).Although all these resins were suitable for the purification of therecombinant adenoviruses, we found Sepharose 4 FF best suitable for ourpurpose based on ability to separate virus from host cell proteins andDNA.

Based upon these and other results (see below), a preferred processaccording to the invention is shown schematically in FIG. 4.

Example 2 Buffer Exchange with High Salt Buffer Improves Virus Process

PER.C6 cells were grown in a 10 L bioreactor and infected withAd5.Adapt.MV-H (with measles virus hemagglutinin as transgene, describedin WO 2004/037294). 2.5 days after infection the cells were lysed with1% Triton® X-100, after 30 minutes Benzonase® (50 units/ml) and MgCl₂were added and incubated for another 30 minutes. The harvest wasclarified over a 0.5 μm Clarigard filter and subsequently by a MillistakDE 30/60 filter (Millipore). The clarified harvest was diluted with anequal volume of 0.6 M NaCl/50 mM HEPES pH 7.5, resulting in a finalconcentration of 0.3 M NaCl. The diluted clarified harvest wasconcentrated 4 times with a 500 kD flatscreen cassette (Biomax 500,Pellicon 2 module Millipore) and subsequently diafiltered with 2diafiltration volumes (DFV) of 0.3 M NaCl/50 mM HEPES pH 7.5; 2 DFV of0.6 M NaCl/50 mM HEPES pH 7.5; 2 DFV of 1.0 M NaCl/50 mM HEPES pH 7.5;and 3 DFV of 0.3 M NaCl/50 mM HEPES pH 7.5. The conductivity of thegenerated permeates was measured and the samples were analysed bySDS-PAGE (FIG. 3). The data showed that histones (M_(w) around 10-20 kDon gels, identity confirmed by mass spectrometry) are passing themembrane pores when the salt concentration of the permeate (andtherefore of the retentate) is in the range of 0.55 and 0.85 M NaCl, orhigher.

A possible explanation is that an electrostatic interaction is brokenunder these salt conditions resulting in release of histones fromcomplexes allowing passage through 500 kD pores. From this experiment itis concluded that introduction of a high salt buffer during the UF/DFstep results in more efficient removal of host cell proteins, especiallyhiston proteins.

Although in this example the cells were lysed first and subsequentlytreated with nuclease (T/B), it is anticipated that the diafiltrationagainst buffer with high salt strength (higher than 0.55 M NaCl, forinstance 1 M NaCl) is also beneficial in the process according to theinvention wherein the nuclease is added to the cells before they arelysed (B/T, see example 1), even though in the B/T process there isalready less histon contamination (see FIG. 2).

Therefore, in a preferred embodiment of the process according to theinvention, the clarified lysate is exchanged against a solutioncomprising 0.8-2.0 M NaCl, preferably about 1 M NaCl, or another saltproviding an equivalent ionic strength (see example 1 and FIG. 4).

Example 3 Removal of Contaminating Nucleoprotein from Recombinant VirusPreparations

Generation of recombinant adenovirus with Ebola nucleoprotein as atransgene is described in example 5. In this example, the purificationof such virus is described.

Experiment 3.1

Ad5dE3x.Adapt.Ebo.NP was purified with the described protocol (seeexample 1, FIG. 4). This method resulted in co-purification of theexpressed Ebola nucleoprotein (NP) transgene with the virus. Filled andfinished product was diluted 1:2 with a buffer containing either 5 MNaCl (final conc 2.5 M), or 2% Tween 20 (final conc 1%) and incubatedfor 1 hr at room temperature before loading onto a Sepharose 4 FFcolumn. The void and retarded fractions were analysed by SDS-PAGE. Theresults (FIG. 5) show that the void fraction contained Adenovirus type 5without contaminating intact NP. Thus far, the results with the highsalt appeared reproducible, whereas those with the detergent were not,and hence high salt is preferred. Optimal conditions for detergenthowever can be tested by varying the detergent used and itsconcentration.

Conclusion: The Ad5dE3x.Adapt.Ebo.NP vector can be purified from theEbola nucleoprotein by incubation in a buffer containing either 2.5 MNaCl or 1% Tween, preferably 2.5 M NaCl, followed by separation on 4 FFsepharose.

Experiment 3.2

Ad5dE3x.Adapt.Ebo.NP was purified with the described protocol (seeexample 1, FIG. 4). Filled and finished product was dialysed with a 10kD membrane against a 50 mM TRIS buffer pH 7.5 containing 1, 2, 3 or 5 MNaCl. The Ad5.Ebo.NP was incubated in these buffers for 2 hours orovernight before loading onto a Sepharose 4 FF column. The void andretarded fractions were analysed by SDS-PAGE. The results show that thevoid fraction contained Adenovirus type 5 with significantly less NP. Asshown in Table 2, the amount of removal of NP relates to the saltconcentration and incubation time.

Conclusion: The Ad5dE3x.Adapt.Ebo.NP vector can be purified from theEbola nucleoprotein by incubation in a buffer containing either 2-5 MNaCl followed by separation on 4 FF sepharose. A longer incubation timeand a higher salt concentration before separation on the 4 FF columnresults in higher purity of the Ad5.Ebo.NP vector (more removal ofnucleoprotein).

Concentrations of 1 M and 1.5 M NaCl are tested with longer incubationtimes (e.g. 2 days, 1 week) according to this same method to find outwhether a longer incubation time may suffice for purification at thesesalt strengths.

Experiment 3.3

The experiment is schematically indicated in FIG. 6. PERC.6 cells weregrown in a 10 L bioreactor and infected with Ad5.dE3x.Adapt.Ebo.NP. 2.5days after infection Benzonase® (50 units/ml) and MgCl₂ were added tothe cell culture, after 10 minutes the cells were lysed with 1% Triton®X-100, and incubated for another 50 minutes. The harvest was clarifiedover a 0.5 μm Clarigard filter and subsequently by a Sartopore 2 filter(0.8/0.45 μm, Sartorius).

The clarified harvest was split in two portions. One portion wasconcentrated 5 times and diafiltered against a buffer containing 5 MNaCl/50 mM Tris pH 7.5 by use of a 0.5 μm hollow fiber (Spectrum). Thisresulted in an increase of trans membrane pressure (TMP) and a reductionin permeate flux while the visual appearance of the retentate turned towhite and less transparent, indicating precipitation of proteins.

The second portion of clarified harvest was concentrated 5 times anddiafiltrated with 6 DFV of 1.0M NaCl/50 mM TRIS pH 7.5 followed by 4 DFVof 0.4 M NaCl/50 mM TRIS pH 7.5 by use of a 0.5 μm hollow fiber(Spectrum). The final retentate was purified over a Sepharose Q-XLcolumn (Amersham).

The Q-XL eluate was also divided into two portions. One portion wasfurther purified and buffer exchanged to 25 mM NaCl/20 mM TRIS/2.5%glycerol (formulation buffer) over a size exclusion column (Sepharose 4FF) in group separation mode (loading of 20% of column volume); this isproduct A in FIG. 6. The other portion was diafiltered against 6 DFV of5 M NaCl/50 mM TRIS pH 7.5 by use of a 0.05 μm hollow fiber (Spectrum):this is further called the high salt virus fraction.

Although the poresizes of the hollow fiber (0.05 μm, about 800 kD) arelarge enough to allow passage of a 100 kD nucleoprotein, nonucleoprotein could be detected in the permeate and no reduction of theamount of nucleoprotein was seen in the retentate. Possibly, theadaptation of one or more TFF parameters (e.g. increase in shear) mayimprove purification of the nucleoprotein. We have further used sizeexclusion (group separation) to achieve this goal.

The high salt virus fraction was again split into two portions: oneportion was directly purified and buffer exchanged to formulation bufferover a size exclusion (group separation) column (product B in FIG. 6),while the second fraction was stored overnight at room temperaturebefore further purifying and buffer exchanging over a size exclusion(group separation) column (product C in FIG. 6).

The three purified bulk lots were analysed to determine purity,infectivity, yield, aggregation and transgene expression.

SDS-PAGE and Western analysis is shown in FIG. 7, and shows that theintact nucleoprotein, as well as NP degradation products (confirmed bymass spectrometry to be NP degradation products), are increasinglyremoved from product A, B and C respectively.

Reverse phase analysis (RP-HPLC)(FIG. 8) shows that the amount of intactnucleoprotein, as well as NP degradation product (eluting at 39minutes), was reduced by introducing the high salt diafiltration stepfrom about 50% (product A) to <5% (product B) and after overnightstorage in 5 M NaCl at room temp even to below the detection limit of 1%(product C). Using both analysis methods, no effect on viral proteinswas observed.

Transgene expression was shown, the infectivity was unaffected and noaggregation occurred (for all three products A, B and C). Apparently,the incubation of the recombinant virus in high salt, even overnight,did not lead to a significant reduction in quality of the virus.

Instead of or in addition to prolonged incubation with high salt andsubsequent size exclusion, a virus suspension that was buffer exchangedwith 5M NaCl was directly filtered using a 0.45 μm hydrophilic filter(Millipac 20).

Unexpectedly, this resulted in a complete removal of NP from the virus(FIG. 9). This experiment is repeated with filters of different poresizes (e.g. 1.2, 1.0, 0.8, 0.22 μm) to determine the range of possiblepore sizes. A 0.8/0.45 μm Sartopore-2 combination is also tested. Thisfiltration step may suitably be combined with a subsequent sizeexclusion chromatography step, and may require shorter incubation timesof the virus in the high salt solution, resulting in a possible savingsin process time.

Conclusions: 1. Diafiltration of the clarified harvest to 5 M NaCl isnot feasible probably due to precipitation of host cell proteins. 2.Incubation of highly purified Ad5dE3x.Adapt.Ebo.NP in 5 M NaCl followedby separation on Sepharose 4 FF or by filtration though a hydrophilicfilter results in purification of Ad5dE3x.Adapt.Ebo.NP from the Ebolanucleoprotein. 3. Prolongation of the incubation step from two hours toovernight results in an even further reduction of residual nucleoproteinfrom <5% to <1%. Filtration through hydrophilic filters may reduce therequired incubation time to obtain the same result.

Hence, it is feasible to remove nucleic acid binding proteins, such asnucleoproteins, e.g. nucleoprotein of Ebolavirus, from recombinantviruses expressing such proteins, by incubation in at least 2M NaCl,preferably at least 3 M NaCl, more preferably 5 M NaCl for purificationpurposes of batches of such viruses.

Example 4 Testing Different Filters for Clarification

PER.C6 cells were grown in a 10 L bioreactor and infected in separateexperiments with different recombinant adenoviruses. 2.5 days afterinfection the cells were lysed with 1% Triton® X-100, after 30 minutesBenzonase® (50 units/ml) and MgCl₂ were added and incubated for another30 minutes. The harvest was used for clarification experiments.

Depth filters, e.g. Clarigard and Polygard had high recovery (>90%) andgood removal of cell debris (microscopic analysis), and were foundsuitable as an initial clarification filter. However the filtrate stilllooked opalescent.

Millistak DE 30/60 and CE50 were found to be less suitable for filteringT/B harvest due to loss of virus (20-45%). In later fractions the yieldincreased but the retention of opalescence decreased, indicating thatthe filter capacity was reached.

Several membrane filters were tested to further clarify the filtrateproduced by Clarigard filtration; e.g. Milligard 0.5 μm, 1.2 μm and1.2/0.22 μm, Durapore 0.22 and 0.65 μm, Lifegard 1.0 and 2.0 μm (allMillipore) and Sartopore-2 0.8/0.45 μm (Sartorius). The Sartopore 2filter was the only filter among those tested that had a good retentionof the opalescence, a high capacity (>20 ml/cm2) as well as a high virusyield (>95%).

The clarified harvest was concentrated and diafiltrated with flatscreenor hollow fiber modules. Several filters were tested to filter the finalretentate, preferably with a 0.45 μm poresize, in order to make thefinal retentate suitable for chromatography, e.g.: Millipack 20,Lifegard 1.0 μm, Polygard 0.6 μm, Intercept Q, Milligard 1.2/0.5 μm.Again the Sartopore 2 filter was the only filter among those tested thathad a good retention of the opalescence, a high capacity as well as ahigh virus yield (>95%).

Although these experiments were done with a T/B harvest, laterexperiments have confirmed the results above for a B/T harvest accordingto the invention, and hence a Sartopore 2 filter gives very good resultswith the methods according to the invention.

Hence, for the clarification in the methods according to the inventionpreferably a combination of 0.8 μm and 0.45 μm filters, preferably aSartopore 2 filter, is used.

Example 5 Generation and Purification of Different RecombinantAdenoviruses

Various recombinant adenoviruses were purified with methods according tothe present invention. Such viruses can for instance be generated byhomologous recombination in the packaging cells of a left-end part(sometimes referred to as ‘adapter-plasmid’, useful for easy cloning ofthe transgene) and a right-end part of the genome according to methodsknown from the art, such as for instance described in EP 0955373, WO03/104467 and WO 2004/001032. The viruses can be propagated in packagingcells known from the art, such as for instance 293 cells, PER.C6™ cells(exemplified by cells deposited at the ECACC under no. 96022940, seeU.S. Pat. No. 5,994,128), or PER.E1B55K cells expressing E1B 55K proteinfrom Ad35 (see U.S. Pat. No. 6,492,169). Construction of somerecombinant adenoviruses that were and are purified according to themethods of the invention is described in this example.

Adenovirus with Ebolavirus Transgenes

Generation of pAdapt.Ebola NP

The gene encoding the Ebola subtype Zaire nucleoprotein was amplified bypolymerase chain reaction using primers; forward 6401 5′ GCA CCG GTG CCGCCA TGG ATT CTC GTC CTC A 3′ (SEQ. ID. NO. 1) and reverse 6401 5′ GCGCTA GCT CAC TGA TGA TGT TGC AG 3, (SEQ. ID. NO. 2) in order to introducerestriction endonuclease recognition sites and a consensus sequence foroptimal translation initiation (Kozak M, 1987, At least six nucleotidespreceding the AUG initiator codon enhance translation in mammaliancells. J Mol Biol. 20: 947-950) for directional cloning in pAdApt™ (seeEP 0955373). PCR reactions were performed in a Biometra T1 or T3 thermalcycler using 10 uM of each primer, 0.75 ul miniprep DNA of VRC6401 (seeWO 03/028632), 1.5 units Pwo DNA polymerase, 5 ul 10×PCR buffer, 0.5 ul20 mM dNTPs using the following conditions: 1 cycle 5′ 94° C., 1′ 50°C., 4′ 72° C., 5 cycles of 1′ 94° C., 1′ 50° C., 4′ 72° C., 20 cycles of1′ 94° C., 1′ 62° C., 4′ 72° C., 1 cycle of 1′ 94° C., 1′ 62° C., 10′72° C. Subsequently the PCR product of the correct size was digestedwith PinA I (Isoschizomer of Age I) and ligated into the pAdApt™ vectordigested with PinA I and Hpa I. After ligating the fragment for 2 hoursat room temperature, 50% of the mixture was transformed to E. coli DH5αT1R cells by heatshock transformation and plated onto LB agar platessupplemented with 50 ug/ml ampicillin. Twenty colonies were picked andgrown overnight at 37° C. in LB supplemented with ampicillin. MiniprepDNA was extracted using the Qiagen miniprep Spin kit as described by themanufacturer. After restriction enzyme analysis with Hind III and Xba Ia correct clones was selected and further checked by DNA sequenceanalysis.

Generation of pAdapt.Ebola GP (Z)

The gene encoding the Ebola subtype Zaire full-length glycoprotein wasamplified by PCR using primers Forward 6001 (5′ CCC AAG CTT GCC GCC ATGGGC GTT ACA GG 3′) (SEQ. ID. NO. 3) and Reverse 6001 (5′ GGC TCT AGA TTACTA AAA GAC AAA TTT GC 3′) (SEQ. ID. NO. 4). PCR reactions wereperformed in a Biometra T1 or T3 thermal cycler using 10 uM of eachprimer, 100 ng and 25 ng DNA of VRC6001 (see-WO 03/028632), 1.5 unitsPwo DNA polymerase, 5 ul 10×PCR buffer, 0.5 ul 20 mM dNTPs using thefollowing conditions: 1 cycle 5′ 94° C., 1′ 55° C., 4′ 72° C., 5 cycles1′ 94° C., 1′ 55° C., 4′ 72° C., 20 cycles 1′ 94° C., 1′ 64° C., 4′ 72°C., 1 cycle 1′ 94° C., 1′ 64° C., 10′ 72° C. Subsequently the PCRproduct of the correct size was digested with Hind III and Xba I andligated into the likewise digested pAdApt™ vector. After ligating thefragment for 2 hours at room temperature, 50% of the mixture wastransformed to E. coli DH5α T1R cells by heatshock transformation andplated onto LB agar plates supplemented with 50 ug/ml ampicillin.Colonies were picked and grown overnight at 37° C. in LB supplementedwith ampicillin. Miniprep DNA was extracted using the Qiagen miniprepSpin kit as described by the manufacturer. After restriction enzymeanalysis with Hind III and Xba I correct clones were selected andfurther checked by DNA sequence analysis.

Generation of pAdapt.Ebola GPdTM(Z) and pAdapt.Ebola GPdTM(S)

Similarly as described above, codon optimized sequences encoding one ofthe Ebola subtypes Zaire and Sudan/Gulu glycoprotein with a deletion ofthe C-terminal 29 amino acids long transmembrane domain (GPdTM(Z), andGPdTM(S), respectively, see also WO 03/028632), were cloned into pAdapt.

Generation of Recombinant Adenoviruses with Ebolavirus Transgenes

The pAdapt plasmids with the different inserts (pAdapt.Ebola NP,pAdapt.Ebola GP (Z), pAdapt.Ebola GPdTM (S), pAdapt.Ebola GPdTM (Z)),were used to form recombinant adenoviruses by homologous recombinationwith plasmids comprising the remainder of the adenovirus type 5 genome(plasmid pWE/Ad.AflII-rITRspΔE3, which is pWE/Ad.AflII-rITRsp (see EP0955373) with a deletion of 1878 bp in the E3 region (XbaI region) wasused for the right end of the adenovirus genome), according to wellknown methods such as for instance described in EP 0955373, resulting inviruses named Ad5dE3x.Adapt.Ebo.NP, Ad5dE3x.Adapt.Ebo.GP(Z),Ad5dE3x.Adapt.Ebo.GPdTM(S) and Ad5dE3x.Adapt.Ebo.GPdTM(Z), respectively.Of course, the transgenes can similarly be cloned in adenovirus vectorsof different serotypes, such as Ad35, to generate recombinant adenovirusderived from those serotypes (see e.g. WO 00/70071).

Adenoviruses with Plasmodium Transgene

Generation of pAdapt.CS.pFalc and pAdapt535.CS.Pfalc

A codon optimized circumsporozoite (CS) gene of Plasmodium falciparumwas synthesized and cloned into pCR-script (Stratagene), giving clone02-659, as described in WO 2004/055187. The CS gene was cloned intopAdapt and pAdapt535 (see WO 2004/001032) for generation of respectivelyrecombinant Ad5 and recombinant Ad35 vectors. Clone 02-659 and bothpAdapt vectors were digested with Hind III and BamH I and joined byligation. After ligating the fragment for 2 hours at room temperature,50% of the mixture was transformed to E. coli DH5α T1R cells byheatshock transformation and plated onto LB agar plates supplementedwith 50 μg/ml ampicillin. Colonies were picked and grown overnight at37° C. in LB supplemented with ampicillin. Miniprep DNA was extractedusing the Qiagen miniprep Spin kit. After restriction enzyme analysiswith Hind III and Xba I correct clones were selected and further checkedby DNA sequence analysis.

Recombinant adenovirus serotype 5 with the P. falciparum CS gene wasgenerated as follows (see for instance EP 0955373; also described in WO2004/055187). pAdapt.CS.Pfalc was digested by PacI restriction enzyme torelease the left-end portion of the Ad genome. PlasmidpWE/Ad.AflII-rITRspΔE3 containing the right-end part of the Ad5 genomehas a deletion of 1878 bp in the E3 region (XbaI deletion), and was alsodigested with PacI. The digested constructs were co-transfected intoPER.C6 cells, such as deposited at the ECACC under number 96022940. Uponhomologous recombination of the overlapping sequences, recombinant virusnamed Ad5ΔE3.CS.Pfalc was formed.

Recombinant adenovirus serotype 35 with the P. falciparum CS gene wasgenerated similarly, but now PacI-digested pAdapt535.CS.Pfalc was usedfor the left-end of the virus genome, and NotI-digestedpWE.Ad35.pIX-rITRΔE3 (see WO 2004/001032) was used for the right-end ofthe virus genome, and both were transfected into PER-E1B55K producercells (having E1B-55K sequences derived from Ad35; cells have beendescribed in U.S. Pat. No. 6,492,169). Upon homologous recombination ofthe overlapping sequences, recombinant virus named Ad35ΔE3.CS.Pfalc wasformed. Of course, it would also be possible to change the E4-orf6protein in the backbone of the Ad35 virus into E4-orf6 of Ad5, to renderit possible to propagate such viruses on packaging cells that expressthe E1B protein of Ad5, such as PER.C6 or 293 cells (see WO 03/104467).

Ad5ΔE3.CS.Pfalc and Ad35ΔE3.CS.Pfalc are purified according to themethods of the present invention.

In addition, an Ad35 vector with the CS gene, based onpAdapt535.CS.Pfalc with an Ad35 backbone was constructed, having adeletion in E3 and further comprising E4-orf6 of Ad5: this vector isfurther referred to as Ad35.CS.

Several Adenovirus vectors were purified with the described process(example 1, FIG. 4): Ad5dE3x.Adapt.Ebo.GPdTM(Z);Ad5dE3x.Adapt.Ebo.GPdTM(S); Ad5dE3x.Adapt.Ebo.NP, andAd5dE3x.Adapt.Empty on a 2 to 20 L scale. The filled and finished (F&F)products were analysed for purity by reverse phase and SDS-PAGE andfound to be purified near homogeneity (except for the presence of theEbola nucleoprotein in the preparations of the vectors having Ebolanucleoprotein as a transgene). The amount of residual host cell DNA wasmeasured by Q-PCR and was below 100 pg DNA/1E11 VP (as shown in Table 1)

Aggregation was measured by optical density measurements at 320 and 260nm, and also by disc centrifugation. None of the batches showedaggregation. Potency was shown in all batches by a VP/IU ratio below 10,and transgene expression was shown in A549 cells.

The final yield ranged from 20-50% dependent on the scale: 2 L: 24-26%(n=2), 10 L: 30-37% (n=3), 20 L: 46% (n=1).

Example 6 Ad35 Purification Using Anion Exchange Chromatography VersusCharged Filters

PER.C6 cells were grown in a stirred tank to cell density of about 1million cells/ml. The cells were infected with the Ad35.CS vector with aMOI of 40. After 4 days of virus production the infected cell culturewas treated with Benzonase and Triton X-100 (B/T method) as described inexample 1. The B/T harvest was clarified as described in example 1. Theclarified harvest was concentrated 5 times by TFF (using a 0.05 μmhollow fiber), and subsequently diafiltered against 10 diafiltrationvolumes of 0.1 M NaCl, 0.05% PS80, 50 mM Tris pH 7.5. The concentratedand diafiltered retentate was filtered over a 0.45 um filter, and loadedonto the capturing column or filter. As a capture step a Q-XL column (3ml column, 15 cm bedheight) or a Sartobind 75 filter (charged filtercontaining anionic groups, Sartorius) were tested. The bound componentswere eluted with a gradient from 0 to 1 M NaCl in a TRIS-based buffer.The elution profile of the charged filter shows an extra peak at thebeginning of the gradient, which is separated from the Ad35 peak. TheAd35 virus peak elutes from the charged filter in a sharper peak at ahigher salt concentration, 0.44 M NaCl (start 0.41, end 0.49 M NaCl)compared to the Q-XL resin, 0.39 M NaCl (start 0.19, end 0.53 M NaCl).The eluted fractions were analysed by SDS-PAGE, HPLC-AEX, disccentrifugation and TCID50.

The extra peak does not behave as intact Ad35 virus particles, whenanalysed by HPLC-AEX chromatography and disc centrifugation (FIG. 11).SDS-PAGE analysis of the chromatography fractions shows the followingresults (FIG. 12): In the flowthrough of both runs no or very lowamounts of proteins are visible. The extra peak from the charged filterchromatogram shows some but not all Ad35 proteins. In the extra peakviral proteins IIIa, V, VI and VII appear to be missing, while viralproteins II, III, IV and 52.55 k are present.

From these analysis data it can be concluded that charged filters canseparate viral proteins from intact viral particles, while Q-XLsepharose cannot. If no separation occurs this will most likely not bedetected by assays to assess purity like RP-HPLC or SDS-PAGE, since allproteins present in the extra peak are also present in the intactvirion. TABLE 1 Reduction of the amount of residual host cell DNA inpurified bulk samples by reversing the T/B to a B/T harvest method. Theharvest was purified on a 2-20 L scale. See example 1 for details. HostCell ng HC harvest DNA VP/ml DNA/ Run vector method ng/ml HPLC-AEX 1E11VP 1 Ad5.MV-H T/B 0.41 5.40E+10 0.78 2 Ad5dE3x.Adapt.Ebo. T/B 4.315.25E+11 0.82 GPdTM (Z) 3 Ad5dE3x.Adapt.Ebo. B/T 0.46 7.80E+11 0.06 NP 4Ad5dE3x.Adapt.Ebo. B/T 0.44 6.80E+11 0.07 NP 5 Ad5dE3x.Adapt.Empty B/T0.40 8.90E+11 0.04 6 Ad5dE3x.Adapt.Ebo. B/T 0.25 4.66E+11 0.05 NP 7Ad5dE3x.Adapt.Ebo. B/T 0.55 6.60E+11 0.08 GPdTM (S) 8 Ad5dE3x.Adapt.Ebo.B/T 0.15 6.60E+11 0.02 GPdTM (Z) 9 Ad353.CS B/T 0.62 5.15E+11 0.12

TABLE 2 NP removal at different ionic strength and after differentincubation times. See example 3 for details. 2 hours Overnight 1 M Nacl− − 2 M Nacl − + 3 M Nacl +/− + 5 M Nacl + ++

1. A method for the purification of a virus from a host cell, saidmethod comprising in the given order the steps of: a) culturing hostcells that are infected with a virus, b) adding nuclease to the cellculture, and c) lysing said host cells to provide a lysate comprisingthe virus.
 2. The method according to claim 1, said method furthercomprising: d) clarification of the lysate.
 3. The method according toclaim 1, said method further comprising: e) purifying the virus with atleast one chromatography step.
 4. The method according to claim 1,wherein said virus is a recombinant adenovirus.
 5. The method A methodaccording to claim 1, wherein the nuclease of step b) is BENZONASE®. 6.The method according to claim 1, wherein step c) of lysing the hostcells is performed with a detergent.
 7. The method according to claim 6,wherein the detergent is TRITON® X-100.
 8. The method according to claim2, wherein step d) comprises depth filtration and membrane filtration.9. The method according to claim 8, wherein the membrane filtration isperformed using a combination of 0.8 μm and 0.45 μm filters.
 10. Themethod according to claim 3, wherein prior to step e) the clarifiedlysate is subjected to ultrafiltration and/or diafiltration.
 11. Themethod according to claim 10, wherein the clarified lysate that issubjected to diafiltration is exchanged against a solution comprising0.8-2.0 M NaCl, preferably about 1 M NaCl, or another salt providing anequivalent ionic strength.
 12. The method according to claim 3, whereinstep e) comprises anion exchange chromatography.
 13. The methodaccording to claim 12, wherein said anion exchange chromatography isperformed using a charged filter comprising anion exchange groups. 14.The method according to claim 3, wherein step e) comprises sizeexclusion chromatography.
 15. The method according to claim 3, whereinstep e) comprises: e,i) anion exchange chromatography, and e,ii) sizeexclusion chromatography.
 16. The method according to claim 15, whereinthe mixture containing the recombinant adenovirus is buffer exchangedwith a solution comprising at least 2 M NaCl, or another salt providingan equivalent ionic strength, between said steps of anion exchangechromatography and size exclusion chromatography.
 17. The methodaccording to claim 2, wherein buffers used in steps d) and subsequentsteps are free of detergent, magnesium_chloride and sucrose.
 18. Amethod for the purification of a virus that is able to lyse host cells,said method comprising the steps of: a) culturing host cells comprisingsaid virus able to lyse host cells, b) harvesting virus following theirrelease into culture fluid without addition of an external lysis factor,characterized in that a nuclease is added to the culture before 95% ofthe host cells has been lysed.
 19. A method for the production of avirus comprising a nucleic acid sequence coding for a nucleoprotein of ahemorrhagic fever virus, comprising the steps of: a) culturing hostcells that have been infected with said virus, b) subjecting saidculture of host cells comprising said virus to lysis of the host cellsto provide a lysate comprising said virus, c) subjecting the virus toanion exchange chromatography, characterized in that after anionexchange chromatography the virus containing mixture is buffer exchangedwith a solution comprising at least 1 M NaCl, or another salt providingan equivalent ionic strength and/or with a solution comprising at least1% of a detergent.
 20. The method according to claim 19, wherein thevirus containing mixture is buffer exchanged at least once with asolution comprising at least 1 M NaCl, or another salt providing anequivalent ionic strength.
 21. The method according to claim 19, whereinsaid virus is a recombinant adenovirus.
 22. The method according toclaim 19, wherein said hemorrhagic fever virus is Ebola_virus.
 23. Themethod according to claim 20, wherein said solution comprises at least1.5 M NaCl, or another salt providing an equivalent ionic strength. 24.The method according to claim 23, wherein said solution comprises atleast 2 M NaCl, or another salt providing an equivalent ionic strength.25. The method according to claim 24, wherein said solution comprises atleast 3 M NaCl, or another salt providing an equivalent ionic strength.26. The method according to claim 25, wherein said solution comprisesabout 5 M NaCl, or another salt providing an equivalent ionic strength.27. The method according to claim 27, further comprising filtering thevirus containing mixture that is buffer exchanged through a hydrophilicfilter with a pore size of 1.2 μm or less.
 28. The method according toclaim 27, wherein said pore size is about 0.45 μm or about 0.22 μm. 29.The method according to claim 19, further comprising subjecting thevirus containing mixture that is buffer exchanged to size exclusionchromatography.
 30. A method for removing free adenovirus proteins froma recombinant adenovirus preparation, comprising the step of: subjectinga recombinant adenovirus preparation comprising free adenovirus proteinsto a charged filter that contains anion exchange groups.
 31. The methodaccording to claim 30, wherein said recombinant adenovirus preparationcomprises a subgroup B recombinant adenovirus.
 32. The method accordingto claim 30, wherein said recombinant adenovirus is an Ad35 recombinantadenovirus.
 33. The method according to claim 2, said method furthercomprising: e) further purifying the virus with at least onechromatography step.
 34. The method according to claim 3, wherein anybuffers used in step e) and subsequent steps are free of detergent,magnesium chloride and sucrose.